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Recombinant ε protein for inhibiting Clostridium perfringens infection and its preparation method and application

A technology for Clostridium perfringens and protein, which is applied in the fields of biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., and can solve problems such as difficulty in increasing the renaturation rate.

Inactive Publication Date: 2020-01-14
CHINA ANIMAL DISEASE CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein denaturation and renaturation is an extremely complicated process. The renaturation conditions of different proteins are different, and the renaturation rate is often difficult to improve.
This is the main constraint limiting its application

Method used

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  • Recombinant ε protein for inhibiting Clostridium perfringens infection and its preparation method and application
  • Recombinant ε protein for inhibiting Clostridium perfringens infection and its preparation method and application
  • Recombinant ε protein for inhibiting Clostridium perfringens infection and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Soluble expression of ε-hisY

[0089] 1. Synthetic genes

[0090] The present application designed three kinds of recombinant ε genes, respectively ε-hisY gene shown in SEQ ID No.1, ε-hisW gene shown in SEQ ID No.3, and pmε-hisW gene shown in SEQ ID No.4.

[0091] Both the ε-hisY gene and the ε-hisW gene encode the protein ε-his shown in SEQ ID No.2. The pmε-hisW gene encodes the protein pmε-hisW shown in SEQ ID No.5. ε-his is a protein obtained by deleting amino acid residues 52-59 of pmε-hisW.

[0092] Synthesize the ε-Y gene shown in the 151-1113th position of SEQ ID No.1 (the protein shown in the 51-370th amino acid residue of encoding SEQID No.2) by chemical synthesis method, SEQ ID No. The epsilon-W gene shown in No. 151-1113 of 3 (coding the protein shown in No. 51-370 amino acid residues of SEQ ID No.2), pmε shown in No. 151-1137 of SEQ ID No.4 -W gene (encodes protein pmε-W represented by amino acid residues 51-378 of SEQ ID No.5).

[0093] 2. C...

Embodiment 2

[0112] Embodiment 2, animal immune protective test of ε-his

[0113] 1. Preparation of anti-Clostridium perfringens vaccine

[0114] The ε-his protein purified by molecular sieves in Example 1 was dissolved in sterile PBS to obtain an ε-his solution with an ε-his concentration of 1000 μg / mL for immunization. The ε-his solution and Freund's adjuvant were mixed in an equal volume of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named the first vaccine. The ε-his solution and incomplete Freund's adjuvant were mixed in an equal volume of 1:1, and emulsified to prepare an oil emulsion vaccine, which was named the second-immune vaccine.

[0115] Take out the B-type Clostridium perfringens virulent strain C58-5 and the D-type Clostridium perfringens virulent strain C60-11 purchased from the China Veterinary Drug Administration. In the ultra-clean workbench, use 75% alcohol cotton ball to carefully wipe the outer wall of the ampoule that preserves the bacteria, t...

Embodiment 3

[0134] Example 3, Optimization of ε-his Induced Expression Conditions

[0135] 1. Optimization of induction temperature and time

[0136] Inoculate BL21(DE3) / pET30a-ε-Y in LB liquid medium containing 50 μg / ml kanamycin (add kanamycin to LB liquid medium until the concentration of kanamycin is 50 μg / ml to obtain culture medium) at 37°C, using a ThermoMaxQ6000 full-temperature shaker at 200rpm to shake and cultivate to OD 600 When the value (the LB liquid medium containing 50 μg / ml kanamycin was used as the blank control) reached 0.6, isopropylthio-β-D-galactoside (IPTG) was added to induce the following six kinds of expression respectively. The first induced expression was induced with 0.75 mM IPTG for 1 hour at 37°C. The second induced expression was induced with 0.75 mM IPTG for 2 hours at 37°C. The third induced expression was induced with 0.75 mM IPTG for 4 hours at 37°C. The fourth induced expression was induced with 0.75 mM IPTG for 5 hours at 37°C. The fifth induced...

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Abstract

The invention discloses recombinant epsilon protein for inhibiting clostridium perfringens infection and a preparation method and application thereof. The recombinant epsilon protein is (a) protein formed by amino acid sequences shown in SEQ ID No.2 or (b) protein formed by amino acid sequences from the 51 site to the 370 site in SEQ ID No.2 or fusion protein obtained by fusing protein tags on carboxyl terminals or / and amino terminals of the (a) protein or the (b) protein. After animals are immune to the recombinant epsilon protein, the animals can generate the high serum antibody level and can resist attack of clostridium perfringens. The recombinant epsilon protein is good in dissolvability and easy to purify and can be taken as a diagnostic antigen or prepared into a monoclonal antibody or used for further studying the protein function and the conformation relationship.

Description

technical field [0001] The invention relates to a recombinant ε protein for inhibiting Clostridium perfringens infection in the field of biotechnology, a preparation method and application thereof. Background technique [0002] Clostridium perfringens (Clostridium Perfringens), also known as Clostridium welchii, is an important zoonotic disease. The pathogen is traumatic gas gangrene and human food poisoning, as well as sheep blight, lamb dysentery, and necrosis of cattle and sheep. It is one of the main pathogens of acute enteritis and cattle and sheep enterotoxemia, which has caused huge economic losses to animal husbandry. The main pathogenic factor of Clostridium perfringens is the exotoxin secreted by it. There are as many as 13 types, among which α, β and ε are the most important exotoxins. According to the different types of exotoxins produced, the perfringens can be Clostridium mycobacterium is divided into five serotypes A, B, C, D, and E. The epsilon toxin is onl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/33C12N15/70A61K39/08A61P31/04
CPCA61K39/08A61K2039/55566C07K14/33C12N15/70C12N2800/101
Inventor 宋晓晖孙雨翟新验赵柏林
Owner CHINA ANIMAL DISEASE CONTROL CENT
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